Annals of Clinical & Laboratory Science, vol. 32, no. 4, 2002
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Serum Oxidant/Antioxidant Status in Patients with Behçet’s Disease Seyithan Taysi,1 Ibrahim Kocer,2 Ramazan Memisogullari,1 and Ahmet Kiziltunc 1 1 Biochemistry and 2 Ophthalmology Departments, Ataturk University Medical School, Erzurum, Turkey Abstract. The aims of this study were to assess whether the increased oxidative stress in affected tissues is reflected by serum lipid peroxidation and to check for alterations in serum levels of extracellular antioxidants and antioxidant enzyme activities in patients with Behçet’s disease (BD). Serum malondialdehyde (MDA) and ceruloplasmin (Cp) levels and CuZn-superoxide dismutase (CuZn-SOD) and glutathione peroxidase (GSH-Px) activities were increased, while serum transferrin (Trf ) levels were diminished in patients with active ocular BD (n=19), inactive ocular BD (n=18), and nonocular BD (n=15), compared to healthy controls (n=20). Serum MDA levels in patients with active ocular BD and nonocular BD were significantly higher than in the inactive ocular BD group. Patients with active ocular BD also had significantly higher serum Cu-Zn SOD activities, compared to the inactive ocular BD. Erythrocyte sedimentation rate (ESR) and serum C-reactive protein (CRP) levels were higher in patients with active ocular BD, inactive ocular BD, and nonocular BD, compared to the control group. In addition, patients with active ocular BD and nonocular BD had significantly higher ESR and serum CRP levels, compared to the inactive ocular BD group. Serum albumin concentrations showed no significant differences among the BD patients and controls. The authors speculate that in BD patients, serum superoxide radicals may be dismutated to H2O2 by increased CuZn-SOD activity and the conversion of H2O2 to hydroxyl radical may be enhanced by iron, owing to diminished serum Trf; these mechanisms may contribute to the increased serum lipid peroxidation. (received 15 June 2002; accepted 8 July 2002)
Key words: Behcet’s disease, ceruloplasmin, transferrin, albumin, lipid peroxidation, free radicals Introduction Behçet’s disease (BD), first described by a Turkish physician, Hulusi Behçet, is a chronic, progressive disorder that affects many systems of the body, including the eye. The origin of this disease is unclear, but immunoregulatory abnormalities have been proposed as pathogenic mechanisms [1-3]. Common manifestations include oral aphthous ulcers, genital ulcers, skin lesions, and uveitis, which may cause blindness. Involvement of the G-I tract, central nervous system, and vascular system is less common but accounts for mortality associated with BD disease. Adult and pediatric cases occur [4,5]. Address correspondence to Seyithan Taysi, M.D., Department of Biochemistry, Ataturk University Medical School, 25240 Erzurum, Turkey; tel 90 442 342 0945; fax 90 442 236 1301; e-mail:
[email protected].
Behçet’s disease is found worldwide, but there are marked geographical differences in disease expression. BD is more frequent in countries along the ancient Silk Route, which extends from eastern Asia to the Mediterranean basin. The incidence of BD cases per 100,000 population varies from 80370 in Turkey to 13.5-20 in Saudi Arabia, Iran, Korea, Japan, China, and Oman [4,5]. Oxygen free radicals and lipid peroxides have been implicated in the pathogenesis of numerous diseases such as diabetes mellitus, cancer, rheumatoid arthritis, systemic lupus erythematosus, infectious diseases, atherosclerosis, and in aging [6-9]. Growing evidence indicates that oxidative stress is increased in BD, owing to overproduction of reactive oxygen species (ROS) and decreased efficiency of antioxidant defenses [10-12]. To control the flux of ROS, aerobic cells have developed their own defense system, the antioxidant
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system, which includes enzymatic and nonenzymatic components [7]. The antioxidant system consists of low molecular weight antioxidant molecules such as glutathione (GSH) and various antioxidant enzymes. For instance, superoxide dismutase (SOD), the first line of defense against oxygen-derived free radicals, catalyses the dismutation of the superoxide anion (O2.-) into H 2 O 2 . Glutathione peroxidase (GSH-Px) is a selenoprotein that reduces lipidic or nonlipidic hydroperoxides as well as H2O2 while oxidizing GSH. Oxidized glutathione (GSSG) is reduced back to GSH by glutathione reductase [13-16]. Albumin (Alb) contains 17 disulphide bridges and has a single remaining cysteine residue that is responsible for the capacity of albumin to neutralize peroxyl radicals. This property is important, since Alb plays a role in transporting free fatty acids in the serum [17]. A major contributor to the antioxidant defence system of human serum is ceruloplasmin (Cp), which acts as an antioxidant by several mechanisms: (a) inhibiting irondependent lipid peroxidation (Lp) and HO . formation from H2O2 by its ferroxidase activity, (b) reacting with and scavenging H2O2 and superoxide anion, and (c) inhibiting copper-induced Lp by binding Cu2+ ions [18]. Cp is a plasma glycoprotein that is synthesized primarily in the liver and secreted into the blood. Cp permits the incorporation of iron into transferrin (Trf ) without the formation of toxic Fe products [19-21]. The process of lipid peroxidation involves oxidative conversion of polyunsaturated fatty acids to products such as malondialdehyde (MDA), which is usually measured as thiobarbituric acid reactive substances (TBARS), or as lipid peroxides [22]. Patients and Methods A total of 52 BD patients (31 men, 21 women; mean age 27.8±5.0 yr; range 18-39 yr) and 20 healthy volunteers (12 men, 8 women, mean age 26.4±5.3 yr; range 19-38 yr) were included in this study. The protocol followed the tenets of the Declaration of Helsinki. All patients had complete ophthalmologic and systemic examination to define ophthalmic and systemic involvement and disease activity.
The diagnostic criteria for Behçet’s Syndrome proposed by the International Study Group for Behçet’s Disease were used for diagnosis [23]. These criteria include a required element of recurrent oral ulceration, plus at least two of the following: recurrent genital ulceration, ocular inflammation, skin lesions, and positive pathergy test (ie, local inflammatory reaction to scratches or intradermal saline injection). BD cases were assigned to 3 groups according to the activity of uveitis and other findings of BD disease. The patients all had oral ulcerations, genital ulcerations, and positive pathergy test either previously or concurrently with the study. If ocular findings such as anterior uveitis, posterior uveitis, cells in vitreous humor on slitlamp examination, or retinal vasculitis were present, the patients were assigned to the “active ocular BD group.” If the patients with ocular findings mentioned above were treated and showed no activity during the past 6 mo, they were assigned to the “inactive ocular BD group.” The “nonocular BD group” consisted of patients without ocular findings at any time. The controls included 20 sex- and age-matched healthy volunteers without systemic or ocular disease. Erythrocyte sedimentation rate (ESR) was determined by the Westergreen method using anticoagulated whole blood. Venous blood was collected in Vacutainers without additives, allowed to clot 30 min at room temperature, and centrifuged at 3000 x g for 5 min to separate serum. Serum samples were stored at -80°C. Hemolysed samples were excluded. Serum MDA was determined by the thiobarbituric acid method [24]. Serum aliquots (0.2 ml) were mixed thoroughly with 0.8 ml of phosphatebuffered saline (pH 7.4) and 0.025 ml of butylated hydroxytoluene solution. After addition of 0.5 ml of 30% trichloroacetic acid, the samples were placed on ice for 2 hr and then centrifuged at 2000 x g at 25°C for 15 min. One ml of supernatant was mixed with 0.075 ml of 0.1 mol/L EDTA and 0.25 ml of 1% thiobarbituric acid in 0.05 N sodium hydroxide. The samples were placed in boiling water for 15 min, cooled to room temperature, and the absorbance was determined at 532 nm. Total thiobarbituric acidreactive substances (TBARS) were expressed as
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Table 1. Results (mean ± SD) of biochemical assays in the patients with Beçhet’s disease (BD) and control subjects.
number of subjects male female age (yr)
Active ocular BD
Inactive ocular BD
Nonocular BD
Healthy Controls
19 12 7 27.6±5.6
18 11 7 27.6±5.5
15 7 7 28.3±4.0
20 12 8 26.4±5.3
395.0±8.6a 153.0±2.5a 91.6±2.2c 46.6±5.8 44.2±10.1c 288.4±37.4c 26.0±7.8c 12.8±3.2c
471.3±10.3c,d 167.1±3.3a 88.0±2.1c 47.0±7.4 41.5±11.0b 281.9±48.8c 33.0±9.1c,e 18.3±5.6c,e
310.7±10.4 125.2±3.8 12.5±1.6 49.9±5.3 32.0±9.0 333.1±57.9 10.9±4.0 2.9±1.2
serum malondialdehyde (MDA)(nmol/dl) 490.0±10.1c,e serum CuZn-SOD (U/dl) 178.0±4.3a,d serum GSH-Px (IU/dl) 87.5±1.7c serum albumin (Alb) (g/L) 47.6±5.5 serum ceruloplasmin (Cp) (mg/dl) 42.4±7.1c serum transferrin (Trf ) (mg/dl) 280.6±60.6c erythrocyte sedimentation rate (ESR) (mm/hr) 35.7±8.4c,f serum C-reactive protein (CRP) (mg/L) 17.3±7.7c,e a p
